a) Field of the Invention
The invention is directed to a process for confocal microscopy in which laser light of different spectral ranges is coupled into a microscope beam path deflected in at least two coordinates and is directed successively with respect to time onto locations of a specimen, wherein the specimen is acted upon, location by location and line by line, by the laser light in at least one plane and an image of the scanned plane is generated from the light reflected and/or emitted by the irradiated locations. The invention is further directed to a laser scanning microscope for carrying out this process.
b) Description of the Related Art
While conventional light microscopy only enables the optical acquisition of one imaging plane, confocal microscopy, as a special further development of light microscopy, offers the possibility of imaging and measuring microstructures also in the Z spatial axis. With light microscopy, it is not possible, for example, to gain an impression of the spatial structure of the rough surface of a specimen at high magnification because only a small area of the specimen can be shown in sharp focus, while details located deep in the surface are imaged in a blurry manner because of the high scattered light component and deficient axial resolution.
In confocal laser scanning microscopy, on the other hand, the scattered light is extensively eliminated and only the structures located in the focal plane of the objective are imaged. If the radiation is focused on different planes, three-dimensional images of a specimen can be calculated from the scanning of these planes which are staggered in the direction of the Z-axis.
For this purpose, a first pinhole is imaged in the object plane so as to be reduced in a punctiform manner using lasers as an illumination source. The punctiform laser beam is moved over the specimen in a raster pattern from location to location and line by line by means of deflecting mirrors. The light reflected and/or emitted by the specimen is focused through the microscope objective onto a second pinhole which is arranged so as to be conjugated with respect to the first pinhole. As a result of the arrangement of these two pinholes, only information from the focal plane reaches one or more detectors which are arranged following the second pinhole.
The scattered light occurring above and below the focus is eliminated by the second pinhole. The information determined by two-dimensional deflection from a plurality of imaging planes located one above the other is stored and subsequently processed to form images.
This principle of confocal laser scanning microscopy is described, for example, in Schroth, xe2x80x9cKonfokale Laser-Scaning-Mikroskopie, eine neue Untersuchungsmethode in der Materialprxc3xcfung [Confocal Laser Scanning Microscopy, a new method of investigation in materials testing]xe2x80x9d, Zeitschrift Materialprxc3xcfung, volume 39 (1997), 6, pages 264 ff.
Further, it is known from xe2x80x9cMitteilungen fxc3xcr Wissenschaft und Technikxe2x80x9d, volume II, no. 1, pages 9-19, June 1995, to use either individual lasers, each having one wavelength, or xe2x80x9cmulti-linexe2x80x9d mixed gas lasers with a plurality of usable wavelengths as illumination source in laser scanning microscopes. This opens up the possibility of utilizing confocal microscopy for fluorescence technique in addition to the classic contrasting processes of bright field, phase contrast and interference contrast. The basis for this consists in that different fluorochromes whose excitation and emission wavelengths lie in different spectral regions allow structures to be shown in a plurality of fluorescence colors simultaneously. Accordingly, depending on the spectral characteristics of different dye molecules, conclusions may be reached about physiological parameters in addition to morphological information. When the confocal microscope is used for fluorometric processes, information can be derived concerning changes in the concentration of ions and molecules. In this connection, other important indicators are those which show a shifting of the excitation and emission spectrum in addition to the intensity dependence and, in this regard, enable a quantification of ion concentrations. Also proposed in this connection is the photobleaching method in which a defined nonuniformity is generated in order to be able to obtain information about the object such as fluidity and diffusion through the dynamics of the equilibrium which is subsequently initiated.
It is known from the above-cited publication to use Ar-Kr lasers for fluorescence excitation in the visible spectral region with lines 488 nm, 568 nm and 647 nm. These lines are combined in a laser beam and supplied to the scanning device via light-conducting fibers. An Ar laser with wavelengths 351 nm and 364 nm is suggested for excitation in the UV range. Coupling into the scanning device is also carried out in this instance via light-conducting fibers.
The processes and arrangements described herein can be utilized for acquiring 3D data records which allow, for example, a reliable correlation of spatial cell structures and tissue structures within a microarchitecture or the localization of a plurality of gene sites in chromosomes in FISH experiments.
However, a disadvantage consists in that the respective specimen is acted upon over the entire scanning region by the laser radiation that is generated in the laser module and coupled into the scanning unit. The entire scanning region is therefore exposed to a relatively high radiation loading which leads to unwanted effects and insufficient results particularly when investigating living organisms.
A further disadvantage consists in that radiation emitted and/or reflected from a determined location on a specimen cannot be detected and evaluated in a definite manner when the specimen is excited with different wavelengths such as those of the above-mentioned laser lines, since the xe2x80x9cbleed-throughxe2x80x9d effect occurs between the individual spectral lines.
On this basis, the primary object of the invention is to further develop a process for laser scanning microscopy of the type described above in such a way that the radiation loading of the specimen is reduced and a more precise image evaluation is achieved.
According to the invention, this object is met in that a change in the spectral composition and/or in the intensity of light is carried out during the deflection of the laser beam from location to location. This is effected either in that the coupling in of individual spectral components or of a plurality of spectral components or the radiation of the light in its entirety is periodically interrupted or in that individual spectral components or a plurality of spectral components are periodically coupled into the microscope beam path additionally, while the deflection of the microscope beam path continues in an uninterrupted manner.
In this way, at least two locations located next to one another on the specimen are acted upon by light with different spectral characteristics and/or by laser radiation of different intensity. By periodically interrupting the coupling in of the laser light during the deflection of the microscope beam path, it is made possible that only selected portions of the image field are acted upon by the laser radiation.
The specimen is protected in that only the areas of the specimen relevant for image evaluation are acted upon by laser radiation of high intensity.
In a preferred construction variant of the process according to the invention, the spectral composition and/or intensity of the laser light is changed during the scanning of a plurality of locations which are located adjacent to one another and thus form a scanning line. In this connection, the deflection can be carried out over the locations of this line repeatedly in the same direction or also bidirectionally. It is provided according to the invention, for example, that the change in the spectral composition or in the intensity is always carried out with reference to the same locations lying adjacent to one another in this line during every scan over the locations in this line, regardless of whether this scan is carried out in the same direction or in the opposite direction, so that the quality of the image evaluation is increased while the energy introduced into the specimen remains limited. In this way, it is achieved at the same time that individual adjacent locations of the specimen can be observed without bleed-through of individual spectral regions into one another.
The different spectral composition of the laser radiation coupled into the microscope beam path is achieved, for example, in that the radiation provided by a plurality of line lasers, e.g., with wavelengths of 633 nm, 568 nm, 543 nm, 514 nm, 488 nm and 458 nm, is coupled in as required or depending on the characteristics of the specimen to be evaluated with an individual wavelength, with a selection of a plurality of individual wavelengths or with all available individual wavelengths. In addition to this radiation in the VIS range, additional wavelengths in the UV range, for example, 351 nm and 364 nm, can be provided for coupling in.
In preferred constructions of the invention, the laser radiation is coupled into the microscope beam path via single-mode fibers so as to maintain polarization. The respective laser lines provided for radiation are advantageously adjusted to a desired brightness with an acousto-optic tunable filter (AOTF) which can also be followed by an acousto-optic modulator (AOM). The respective laser wavelength is adapted to the microscope objective placed in the beam path for both the UV and the VIS region by variable beam collimation.
A further preferred construction of the process according to the invention consists in that the light reflected and/or emitted by every individual irradiated location of the specimen is evaluated with respect to its spectral characteristics and intensity, wherein the evaluation is carried out synchronously with the irradiation of the same location and while taking into consideration the spectral composition and/or intensity of the laser light by which this location is irradiated. This makes it possible to evaluate the scanned portion of the specimen with respect to individual locations, which leads to a very high resolution and to the highest possible accuracy in the evaluation of the image.
It also lies within the framework of the invention that the laser light reflected and/or emitted by every individual irradiated location is detected with a plurality of detection channels, wherein the individual detection channels are arranged for receiving different spectral components. This provides very good conditions for the examination of multifluorescence specimens, and identical optical sections can be generated via every detection channel with simultaneous reception of multifluorescence specimens.
In this connection, it is provided according to the invention that the spectral composition and/or the intensity of the laser light which is coupled into the microscope beam path corresponds to the excitation radiation of a fluorescence dye contained in the specimen or applied to the specimen and the individual detection channels are configured for the reception of the emission radiation proceeding from the fluorescence dye. This makes it possible to generate laser light for the excitation of different fluorescence dye and to draw conclusions from the detection concerning the distribution of these fluorescence dyes on or in the specimen.
Another very preferable construction of the invention consists in that an evaluation of the spectral composition and/or of the intensity of the coupled in laser light is carried out in a continuous manner and the evaluation findings for the laser radiation directed to a determined location are mathematically linked with the evaluation findings for the light reflected and/or emitted by this location. As a result of this link, for example, the deflection position of the microscope beam path for two adjacent locations can be determined according to the coordinates x, y, z for which differences in the spectral characteristics of the light reflected and/or emitted from these locations which go beyond a predetermined threshold value can be detected during evaluation, wherein, based on these differences, conclusions can be reached concerning the presence of an optical boundary layer between these two locations. These deflection positions are stored, according to the invention, and taken as a basis for the calculation of surface areas and/or volumes enclosed by optical boundary layers within the specimen.
Further, with the deflection positions which are obtained and stored in this way, it is possible to determine and preset adjustment signals for the spectral composition and/or the intensity of the laser light for irradiation of these locations during a subsequent scan cycle, so that an automatic optimization is achieved in the image evaluation while taking into account the optical characteristics of the specimen and of the fluorescence dye.
In particular, the process according to the invention can be used in an advantageous manner for photobleaching, as it is called. In this connection, a selected area of a specimen can be acted upon initially by a relatively high radiation intensity during scanning, thereby initiating a bleaching process. With the scan cycles following immediately thereafter, the reactions taking place are optically detected and evaluated, wherein information can be obtained about the dynamic processes such as diffusion and transport processes taking place in the specimen substance immediately after the bleaching process.
For this purpose, the scanning must be carried out with a very high time resolution, which is achieved, according to the invention, by switching between different intensities and different spectral compositions of the light impinging on individual locations of the specimen, wherein this switching is carried out with sufficient speed in a synchronous manner with respect to the deflection of the beam.
The fast switching between different intensities and different spectral compositions of the laser radiation is carried out with an acousto-optic tunable filter (AOF) which, in an analogous but substantially faster manner, takes over the function of different filters which can be substituted for one another in the beam path and which, further, can also modulate the intensity of individual laser lines or optional combinations of lines in a highly dynamic manner with respect to time.
The manner of operation and application of the AOTF is thoroughly described, for example, in String, Kenneth, R., xe2x80x9cWavelength Selection for Illumination in Fluorescence Microscopyxe2x80x9d, NIH, LKEM, Building 10/6N309, Bethesda, MD 20892, April 1993. Further, concrete application examples for the AOTF are described in U.S. Pat. Nos. 5,444,528, 5,377,003 and 5,216,484.
The synchronization in time between the driving of the AOTF for modulation of the laser radiation and the driving of the scanning device for beam deflection is achieved in that determined control signals for the AOTF are correlated with the control signals supplied to the scanning device by the driving device. Thus, the scanning device and AOTF are always driven synchronously, i.e., the control pulses for the AOTF are, with respect to time, always added to the output of a control pulse for the scanning device.
On the other hand, this means that a characteristic intensity and/or spectral composition of the light can be assigned to every deflection position and accordingly to every location of the specimen.
In this respect, the circuit arrangements for executing the process are optimized by the AOTF with respect to very short transit times of the control pulses from output to switching of beam modulation. These transit times are in the range of  less than 10 ms. In a variant of the process, when controlling the AOTF or the scanning device, rate action times or lead times are calculated beforehand for switching the intensity and spectral composition and/or for deflection, so that precisely the intended location is also irradiated with the intended radiation intensity and spectral composition.
The invention is further directed to a laser scanning microscope for carrying out the process steps described above, with a laser module for generating laser light with different selectable spectral components, with single-mode fibers for coupling the laser light into the microscope beam path, with a scanning device which deflects in at least two dimensions, with a microscope objective which focuses the laser light on a specimen, with a plurality of detectors for the reception of different spectral components of the light reflected and/or emitted by the specimen, and with an evaluation circuit which is connected subsequent to the outputs of the detectors.
In a laser scanning microscope of this kind, according to the invention, a plurality of individually controllable single-wavelength and/or multiple-wavelength lasers are provided in the laser module, the laser module is followed by a beam combiner, an acousto-optic tunable filter (AOTF) and/or an acousto-optic modulator (AOM), the single-mode fiber is followed by collimating optics whose distance from the respective end of the fiber can be changed and which are coupled with drivable adjusting devices. Photomultipliers (PMT) are provided as detectors, each of which is associated with a reflection band or emission band and accordingly with a detection channel. Filters and/or color splitters which are arranged on splitter wheels and which can be substituted for one another by rotating the splitter wheels are provided for branching the radiation proceeding from the specimen into individual detection channels, wherein every splitter wheel is likewise coupled with a controllable adjusting device. Further, the control inputs of the laser module, AOTF, AOM, scanning device and adjusting devices for the splitter wheels and collimating optics are connected with the outputs of the evaluation circuit.
In a construction variant of the laser scanning microscope, the microscope beam path directed on the specimen is branched and one of the branches is directed to an optoelectronic receiver whose output is likewise connected with the driving unit.
Further, it is provided in a preferred construction variant that a mathematical linking of the output signals of the optoelectronic receiver with the output signals of the PMT and/or with the deflection signals for the scanning device is carried out in the evaluation circuit, wherein optimized adjusting signals for the laser module, AOTF, AOM, scanning device and for the adjusting device are made available at the output of the evaluation circuit.
The invention will be explained more fully hereinafter with reference to an embodiment example.